Structures of the two 3D domain‐swapped RNase A trimers

Liu, Yanshun; Gotte, Giovanni; Libonati, Massimo; Eisenberg, David

doi:10.1110/ps.36602

Cited by 118 publications

(120 citation statements)

References 38 publications

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“…2A) (26). This oligomerization state is consistent with gel filtration analysis of SpHCS, which elutes as a ϳ110-kDa species during purification (data not shown).…”

Section: Volume 284 • Number 51 • December 18 2009supporting

confidence: 85%

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Bulfer¹,

Scott²,

Couture³

et al. 2009

Journal of Biological Chemistry

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Homocitrate synthase (HCS) catalyzes the first and committed step in lysine biosynthesis in many fungi and certain Archaea and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N-and C-terminal domains of each monomer. The N-terminal catalytic domain is composed of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed ␣/␤ topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain occludes the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Mutations of the active site residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity in vitro and in vivo. Together, these results yield new insights into the structure and catalytic mechanism of HCSs and furnish a platform for developing HCSselective inhibitors.

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“…2A) (26). This oligomerization state is consistent with gel filtration analysis of SpHCS, which elutes as a ϳ110-kDa species during purification (data not shown).…”

Section: Volume 284 • Number 51 • December 18 2009supporting

confidence: 85%

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Bulfer¹,

Scott²,

Couture³

et al. 2009

Journal of Biological Chemistry

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“…Three-dimensional domain swapping has been proposed as a general mechanism for the self-association of proteins (21,22). The ΔN6β2m dimer we trapped with Nb24 meets all common properties of domain-swapped oligomers (23). First, only one small C-terminal segment of the protein (the rest retaining the native-like structure) participates in the oligomerization, without disrupting the core of the protein fold.…”

Section: Discussionmentioning

confidence: 96%

Atomic structure of a nanobody-trapped domain-swapped dimer of an amyloidogenic β2-microglobulin variant

Domanska

Vanderhaegen

Srinivasan

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

113

120

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Atomic-level structural investigation of the key conformational intermediates of amyloidogenesis remains a challenge. Here we demonstrate the utility of nanobodies to trap and characterize intermediates of β2-microglobulin (β2m) amyloidogenesis by X-ray crystallography. For this purpose, we selected five single domain antibodies that block the fibrillogenesis of a proteolytic amyloidogenic fragment of β2m (ΔN6β2m). The crystal structure of ΔN6β2m in complex with one of these nanobodies (Nb24) identifies domain swapping as a plausible mechanism of self-association of this amyloidogenic protein. In the swapped dimer, two extended hinge loops-corresponding to the heptapetide NHVTLSQ that forms amyloid in isolation-are unmasked and fold into a new twostranded antiparallel β-sheet. The β-strands of this sheet are prone to self-associate and stack perpendicular to the direction of the strands to build large intermolecular β-sheets that run parallel to the axis of growing oligomers, providing an elongation mechanism by self-templated growth.crystallization chaperones | amyloid fibrils | prefibrillar intermediates | dialysis-related amyloidosis P eptides and proteins exhibit a common tendency to assemble into highly ordered fibrillar aggregates, whose formation proceeds in a nucleation-dependent manner (1, 2). The full elucidation of the aggregation process requires the identification of all the conformational states and oligomeric structures adopted by the polypeptide chain. Atomic-level structural investigation of the key conformational intermediates of amyloidogenesis remains a challenge. This is due to the nature of the process, which may be described as a dynamic equilibrium between diverse structural species. These intermediates have dissimilar sizes and occur in very uneven amounts and time frames. Fibril formation in vivo usually takes several years, and the intermediate species are short living and highly unstable (2). Here we demonstrate the utility of heavy chain only antibodies derived from camel (3, 4) for the structural investigation of prefibrillar intermediates of β2-microglobulin (β2m) amyloidosis. The antigen-binding site of these antibodies consists of a single domain, referred to as VHH or nanobody (Nb) (4).β2m is a 99-residue soluble protein that adopts the classical seven-stranded β-sandwich immunoglobulin fold and is expressed as a key component of the major histocompatibility class I complex (MHC-I) on the cell surface of all nucleated cells (5, 6). In healthy individuals, excess β2m is degraded and excreted from the bloodstream by the kidney. In patients suffering from renal failure, the β2m concentration increases up to 60-fold (7) leading to the formation of insoluble amyloid fibrils and causing dialysisrelated amyloidosis (DRA) (8). In amyloid deposits extracted from DRA patients, up to 25-30% of the constituting β2m is truncated and lacks the six N-terminal amino acids (ΔN6β2m) (9, 10). The ΔN6-truncated form of β2m readily aggregates and fibrillates at neutral pH (10, 11).The identification...

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“…Similarly, the same single C-terminal helix was swapped in both dimeric and trimeric cyt c. The domain-swapped dimeric structure of serpin was recently solved, and successive domain swapping of a single domain for polymerization has been suggested from the dimeric structure (14). For higher-order oligomers, domain swapping in RNase A has been suggested but N and C domains both swap and form various oligomers (27). However, successive domain swapping has been suggested as a mechanism for forming fibrils of RNase A (3D domain-swapped zipper-spine model) (12,28).…”

Section: Discussionmentioning

confidence: 99%

Cytochrome c polymerization by successive domain swapping at the C-terminal helix

Hirota

Hattori²,

Nagao³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

157

245

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Cytochrome c (cyt c) is a stable protein that functions in a monomeric state as an electron donor for cytochrome c oxidase. It is also released to the cytosol when permeabilization of the mitochondrial outer membrane occurs at the early stage of apoptosis. For nearly half a century, it has been known that cyt c forms polymers, but the polymerization mechanism remains unknown. We found that cyt c forms polymers by successive domain swapping, where the C-terminal helix is displaced from its original position in the monomer and Met-heme coordination is perturbed significantly. In the crystal structures of dimeric and trimeric cyt c, the C-terminal helices are replaced by the corresponding domain of other cyt c molecules and Met80 is dissociated from the heme. The solution structures of dimeric, trimeric, and tetrameric cyt c were linear based on small-angle X-ray scattering measurements, where the trimeric linear structure shifted toward the cyclic structure by addition of PEG and ðNH 4 Þ 2 HPO 4 . The absorption and CD spectra of high-order oligomers (∼40 mer) were similar to those of dimeric and trimeric cyt c but different from those of monomeric cyt c. For dimeric, trimeric, and tetrameric cyt c, the ΔH of the oligomer dissociation to monomers was estimated to be about −20 kcal∕mol per protomer unit, where Met-heme coordination appears to contribute largely to ΔH. The present results suggest that cyt c polymerization occurs by successive domain swapping, which may be a common mechanism of protein polymerization.dimer | trimer | protein polymer

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Structures of the two 3D domain‐swapped RNase A trimers

Cited by 118 publications

References 38 publications

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Atomic structure of a nanobody-trapped domain-swapped dimer of an amyloidogenic β2-microglobulin variant

Cytochrome c polymerization by successive domain swapping at the C-terminal helix

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